Potential biomarkers and therapeutic targets of idiopathic pulmonary arterial hypertension

Abstract Background Peripheral blood mononuclear cells (PBMCs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). However, the specific roles of PBMCs in the development and progression of idiopathic PAH (IPAH) have not been fully understood. Methods Here, differentially expressed genes (DEGs) of PBMCs or lung tissues between IPAH patients and healthy controls were identified via bioinformatics analysis of Gene Expression Omnibus (GEO) datasets GSE33463 and GSE48149, respectively. Subsequently, extensive target prediction and network analysis were performed to assess protein–protein interaction (PPI) networks, Gene Ontology (GO) terms, and pathway enrichment for DEGs. Co‐expressed DEGs between PBMCs and lung tissues coupled with corresponding predicted miRNAs involved in PAH were also assessed. We identified 251 DEGs in PBMCs and 151 DEGs in lung tissue samples from IPAH. PDK4, RBPMS2, and PDE5A expression were altered in both PBMCs and lung tissues from IPAH patients compared to healthy control. Results CXCL8, JUN, TLR8, IL1B, and TLR7 could be implicated as the hub genes in PBMCs, whereas ENO1, STAT1, CXCL10, GPI, and IRF1 in lung tissues. Finally, co‐expressed DEGs of PDK4, RBPMS2, and PDE5A coupled with corresponding predicted miRNAs, especially miR‐103a‐3p, miR‐185‐5p, and miR‐515‐5p, are significantly associated with IPAH. Conclusion Our findings collectively suggest that the expression levels of PDK4, RBPMS2, and PDE5A in PBMCs are associated with the expression of these genes in lung tissues. Thus, these molecules may serve as potential circulating biomarkers and/or possible therapeutic targets for IPAH.


| INTRODUCTION
Idiopathic Pulmonary Arterial Hypertension (IPAH) is a rare and progressive disease with a poor prognosis, characterized by a progressive increase of pulmonary artery (PA) pressure and pulmonary vascular resistance (PVR) (Keating, 2016;Lau et al., 2017;Maron & Diagnosis, 2016). Although the mechanisms of IPAH remain unknown, there is growing evidence indicating that peripheral blood mononuclear cells (PBMCs) contribute to the pathogenesis of IPAH (Cheadle et al., 2012;Hoffmann et al., 2016;Risbano et al., 2010;Sarrion et al., 2015). Circulating PBMCs include lymphocytes, monocytes, stem cells, and other cell types. Compared to lung tissues, PBMCs can be collected from a broader range of patients; thus, PBMCs are used to study biomarkers for various diseases (Baine et al., 2012;Castaldo et al., 2019;Goleva et al., 2019;Guo et al., 2019;Yang et al., 2016). Lung tissues contain vessels and provide the microenvironment for pulmonary vascular cells, which are pathologically changed in pulmonary hypertension.
The gene expression analysis in lung homogenates may provide insight into genes involved in vascular remodeling in IPAH patients (Hoffmann et al., 2016;Yuan et al., 2016). However, lung tissues are not available from most of IPAH patients. Numerous studies suggest that PBMCs infiltrate lung tissues and play an important role in the pathogenesis of PH (Cheadle et al., 2012;Chesne et al., 2014;Risbano et al., 2010;Sarrion et al., 2015). Elevated numbers of inflammatory cells have been observed in the lungs of patients with IPAH. The recruited inflammatory cells can release mediators to directly change the vascular microenvironment, promoting the proliferation of pulmonary smooth muscle cells and recruiting more circulating inflammatory cells, further aggravating the progress of the disease (Hassoun et al., 2009;Hoffmann et al., 2011;Marsh et al., 2018;Perros et al., 2007;Pienn et al., 2018;Savai et al., 2012). PBMCs play a critical role in activating the innate immune response, which can migrate into the lung tissue (Cheadle et al., 2012;Foris et al., 2016;Khan & Kaihara, 2019;Lenna et al., 2013;Yamamoto, 2011;Yeager et al., 2012). However, the mechanism of how circulating inflammatory cells is recruited to lung tissues and what relations between PBMCs and lung tissues are unknown. To our knowledge, high-throughput technologies such as microarray expression profiles of PBMCs and lung tissues have provided powerful weapons for the diagnosis and prognosis of IPAH patients. Therefore, to make full use of expression profiles data of PBMCs and lung tissues from IPAH patients and better understand the pathogenesis of IPAH, we utilized bioinformatics analysis to explore hub genes and the critical mechanism of IPAH.
Here, we hypothesized that transcriptome changes between PBMCs and lung tissues contribute to the recruitment of inflammation cells, the changes of the vascular microenvironment, and pulmonary vascular remodeling. Thus, in this study, we conducted a bioinformatics analysis for the transcriptome in PBMCs and lung tissues between IPAH patients and healthy control to investigate the contribution of recruited immune cells in lung tissues to the development of IPAH with the hope of identifying potential biomarkers and therapeutic targets for IPAH.

| Sources of the gene data
GSE33463 and GSE48149 datasets were downloaded from Gene Expression Omnibus (GEO) database (http://www. ncbi.nlm.nih.gov/geo/), and the detection platforms for both GSE33463 and GSE48149 datasets were Illumina human V3.0 Expression BeadChip (Edgar et al., 2002). The gene expression profiles of PBMCs from 30 IPAH patients were compared to 41 healthy volunteers. The gene expression profiles of lung tissues are available from 17 donors who underwent lung transplantation (Table 1). These previous studies can provide insightful viewpoints concerning the epidemiological characteristics and phenotyping data related to IPAH (Cheadle et al., 2012;Hsu et al., 2011). They were both used to explore the differentially expressed genes between IPAH patients and healthy controls. The workflow of this study is illustrated in Figure 1. 2.2 | Identification of the differentially expressed genes R packages of "Illumina" and "limma" (http://www.bioco nduct or.org/packa ges/relea se/bioc/html/limma.html), provided by a Bioconductor project, were applied to assess GSE33463 and GSE48149 RAW datasets. We used in lung tissues. Thus, these molecules may serve as potential circulating biomarkers and/or possible therapeutic targets for IPAH.

K E Y W O R D S
biomarker, gene analysis, GEO, IPAH background correction, quantile normalization, probe summarization, and log2-transformation to create a robust multi-array average (RMA), a log-transformed perfect match, and a mismatch probe (PM and MM) methods. The Benjamin-Hochberg method was used to adjust original p-values, and the false discovery rate (FDR) procedure was used to calculate fold-changes (FC). Gene expression values of 2 |log2 FC| >2 and adjusted p < 0.05 were used for DEGs of PBMCs; 2 |log2 FC| >1.5 and adjusted p < 0.05 were used to identify DEGs of lung tissues. Then, two Volcano plots were adopted to visualize the expression differences by R 3.5.0. Additionally, we calculated and made Venn diagrams for co-DEGs for the two datasets.

| Functional analysis
The PBMCs-DEGs and lung-DEGs were subjected for functional annotation and Gene Ontology/ Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) enrichment analysis for annotation, visualization, and integrated discovery of bioinformatics resources by DAVID (http:// david.abcc.ncifc rf.gov/). GO terms and KEGG maps of biological functions associated with a p < 0.05 were significantly enriched. Besides, we presented different biofunctions of PBMC-DEGs and lung-DEGs in biological processes, molecular functions, and cellular components from DAVID, respectively.

| Identification of protein-protein interaction (PPI) networks of DEGs
PPI networks of PBMCs-DEGs and lung-DEGs were analyzed using the search tool for retrieving interacting genes (STRING database, V10.5; http://strin g-db.org/) that can predict protein functional associations and proteinprotein interactions. Subsequently, Cytoscape software (V3.5.1; http://cytos cape.org/) was applied to analyze biological networks and node degrees after downloading the analytic results of the STRING database.

| RESULTS IDENTIFICATION OF DEGS
As indicated in Figure 2a, we found 251 DEGs in PBMCs of IPAH patients compared to healthy controls, including 110 downregulated genes and 141 upregulated genes. We found 151 DEGs in IPAH patients compared with the control population, including 73 downregulated and 78 upregulated genes in the lung tissues. The Volcano plots in Figure 2b illustrated the expression differences. And we identified six co-expressed DEGs based on the DEG overlaps between the two datasets ( Figure 3). As indicated in Table 2, PDK4, RBPMS2, and PDE5A were upregulated in both PBMC and lung tissues from IPAH patients compared to healthy control. TNFAIP3, CCL3, and PHLDA1 were found to be upregulated in lung tissue but downregulated in PBMCs. T A B L E 1 Demographic and clinical characteristics of the patients with IPAH

| Functional GO terms and pathway enrichment analyses
The functional GO terms enrichment was performed to reveal IPAH-related GO categories such as biological processes, molecular functions, cellular components, and KEGG. For biological processes, we identified that the common between PBMCs with the lung tissues is apoptotic progress. For the cellular components, we found it were cytosol and mitochondrion. Their differentially expressed genes have common molecular functions in protein binding, protein homodimerization activity.
The top 10 GO terms of PBMCs-IPAH are shown in Table 3. For DEGs in PBMCs-IPAH, the most enriched GO terms of biological processes were associated with oxygen transport, blood coagulation, and apoptotic process. In the cellular component category, enriched GO terms were mainly associated with hemoglobin complex, platelet alpha granule membrane, and mitochondrion. Besides, GO terms enriched for DEGs in PBMCs-IPAH included oxygen transport activity, protein binding, and oxygen binding in the molecular function category. To explore the significant enrichment of DEGs of PBMCs-IPAH in pathway terms, we performed pathway F I G U R E 1 Workflow of this study. DEGs: differentially expressed genes; IPAH: idiopathic pulmonary arterial hypertension; PBMCs: peripheral blood mononuclear cells annotation of DEGs and obtained DEGs involved in all pathway terms. The DEGs of PBMCs-IPAH were enriched mainly in cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, and the chemokine signaling pathway. It is reported that oxygen transport activity plays a vital role in the vascular tone of pulmonary artery smooth muscle cells and hypoxia stimulates vasoconstriction of the pulmonary vascular (Wu et al., 2021). Cytokine-cytokine receptor interaction, chemokine signaling pathway, and Toll-like receptor signaling pathway are known drivers to pulmonary vascular remodeling and established PH (Farkas et al., 2019;Xiao et al., 2020). NF-kappa B signaling pathway has been reported as a therapeutic target for PH (Liu et al., 2020;Shi et al., 2018;Zhang et al., 2019;Zuo et al., 2020).  The top 10 GO terms of lung-IPAH were performed and shown in Table 4. For DEGs in lung-IPAH, the most enriched GO terms of the biological process were associated with an apoptotic process, erythrocyte differentiation, DNA metabolic processes, regulation of growth, and metabolic process. In the cellular component category, enriched GO terms were mainly associated with hemoglobin complex, platelet alpha granule membrane, and cytosol. Furthermore, in the molecular function category, GO terms enriched for DEGs in lung-IPAH included metabolic pathways, biosynthesis of amino acids, lysosome, and carbon metabolism. To explore the significant enrichment of DEGs of lung-IPAH in pathway T A B L E 4 Gene ontology (GO) and pathway enrichment analysis of lung-DEGs terms, we performed pathway annotation of DEGs and obtained DEGs involved in all pathway terms in Table 4. The DEGs of lung-IPAH were enriched mainly with metabolic pathways, biosynthesis of antibiotics, biosynthesis of amino acids, and lysosome (Table 4). A recent 4-month, open-label study also showed that decreasing PDH(a metabolic-related molecular) has reduced mean PAP and PVR and improved functional capacity in IPAH patients with SIRT3 and UCP2 variants (Spiekerkoetter et al.,). Metabolic abnormalities cause PAH or are secondary to pulmonary vascular disease and subsequent RV failure (Paulin & Michelakis, 2014).

| DISCUSSION
IPAH is a progressive disorder characterized by progressive pulmonary arterial narrowing (Keating, 2016;Lau et al., 2017;Maron & Diagnosis, 2016). Although the pathogenesis of IPAH remains still unclear, increasing lines of evidence suggest that PBMCs may be involved in the pathogenesis of IPAH. It has been reported that PBMCs changes would be reflected by specific transcriptional changes in these cells in PAH patients (Cheadle et al., 2012;Hoffmann et al., 2016;Sarrion et al., 2015). Endothelial progenitor cells, which belong to PBMCs, have been accumulating in the lung upon PH-induction (Yan et al., 2016). Moreover, bone marrow transplantation from PH mice to healthy mice increased the PA pressure in healthy mice (Levy et al., 2019;Nikolic & Yu, 2016). These may suggests that the changes in the PBMC genes might be the cause and effect of IPAH. Thus, we sought to explore the possible association between F I G U R E 4 PPI network. PPI networks from a and b were constructed using the STRING database for DEGs related to PBMCs and lung tissues, respectively (threshold>0.5). a. CXCL8, JUN, and TLR8 could be implicated as the hub genes in PBMCs. b.ENO1, STAT1, and CXCL10 could be implicated as the hub genes in lung tissues PBMCs and lung tissues in IPAH with the hope of identifying potential biomarkers or novel therapeutic targets for IPAH.
For DEGs in PBMCs-IPAH, the most enriched GO terms of biological processes and molecular functions were associated with the oxygen transport activity and oxygen binding. Given the fact that the lung is the major organ for oxygen transport, our findings might suggest a possible relationship between PBMCs and lung tissues concerning oxygen transportation. Some studies have shown that in IPAH patients, due to increased PVR, the right ventricle's compensation is not enough (decreased cardiac output) to meet the demand for oxygen transport, resulting in inefficient lung gas exchange, and low perfusion (Tang et al., 2017).
Apart from the genes that are not consistent with upregulation and downregulation, there still remain four genes altered in both PBMCs and lung tissues from IPAH patients compared to healthy controls, including PDK4, RBPMS2, and PDE5A, which are all upregulated in IPAH patients.
Among these genes, PDE5A has been used as a target for IPAH therapy, including drug such as sildenafil, tadalafil, vardendafil, and so on. PDE5A is a phosphodiesterase specifically binding to cGMP, which is the main enzyme that metabolizes cGMP. Previous studies on PH animal models and PASMC from IPAH patients have shown that PDE5A participates in the excessive proliferation of PASMC. Wharton et al. demonstrated that PDE5A inhibition decreases DNA synthesis via increasing cellular cGMP level and stimulated the apoptosis of human PASMC (Lan et al., 2018;Osinski et al., 2001;Rai et al., 2018;Wharton et al., 2005).
PDK4, a gene coding for an enzyme that suppresses the mitochondrial activity in favor of glycolysis. Yuan K et al. found that increased PDK4 is associated with PAH pericyte hyperproliferation and reduced endothelial-pericyte interactions (Yuan et al., 2016). Piao et al. showed that pyruvate dehydrogenase kinase (PDK) is activated in the right ventricular hypertrophy (RVH), causing an increase in glycolysis relative to glucose oxidation that impairs the right ventricular function (Piao et al., 2013). However, another DEG, RBPMS2, to the best of our knowledge, has not been reported to be associated with IPAH. Several studies have shown that RBPMS2 is related to the plasticity of the smooth muscle cells. Akerberg et al. have demonstrated that the proteins encoded by RBPMS2 may play a key role in regulating the cardiomyocyte differentiation, proliferation, survival, and/ or contractility (Kaufman et al., 2018). Sagnol et al. demonstrated that RBPMS2 is related to stomach smooth muscle development and plasticity. The excessive proliferation of vascular smooth muscle is one of the main pathological changes of PH (Sagnol et al., 2016), the relevant mechanisms of RBPMS2 in PAH development need to be further explored.
These findings suggest that the expression of PDK4, RBPMS2, and PDE5A in PBMCs could predict the expression levels of these genes in lung tissues and might serve T A B L E 5 The Genomes (KEGG) pathways enrichment among predicted miRNAs and co-DEGs as circulating potential biomarkers and/or therapeutic targets for IPAH patients. Further evaluation is ongoing in our group to provide more solid evidence to determine whether these DEGs could serve as biomarkers and/or therapeutic targets for IPAH.

| CONCLUSION
These findings demonstrate the expression of PDK4, RBPMS2, and PDE5A in PBMCs could predict the expression levels of these genes in lung tissue and might serve as circulating biomarkers for IPAH.

| Limitation
The clinical characteristics of patients were collected from the GEO database. The fundamental limitation is the lack of ethnic or geographic diversity in our samples, which may affect the reliability of the results. In addition, since the GEO dataset of PBMCs or lung tissues of IPAH are very limited, and the datasets belonging to the same probe-type are even less, we selected two datasets with the largest sample size to analyze the differentially expressed genes. Further studies with larger sample sizes, more diverse ethnicities, and less heterogeneity are warranted to test our hypothesis.